A kind of split ring steroid compound and its preparation method and application

Abstract

The application discloses a kind of split ring steroids and its preparation and application, by in vitro activity test evaluation, the compound of the present application can significantly inhibit interleukin 6 (IL-6) and interleukin 1 beta (IL-1 beta) release induced by lipopolysaccharide in macrophage RAW 264.7 in vitro, indicating that it has good anti-inflammatory activity.Therefore, it can be used for preparing new anti-inflammatory drugs.The present application provides a new source for the development of new anti-inflammatory drugs.

Description

(I) Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a novel split-ring steroidal compound isolated from *Hypericum chinense* fermentation powder (the contents of Bailin capsules), its preparation method, and its application in anti-inflammatory properties. (II) Background Technology

[0002] Cordyceps sinensis (Berk.) Sacc. is a traditional and precious Chinese medicinal herb, a complex formed by the Cordyceps fungus parasitizing the overwintering larvae of the ghost moth *Hepialus armoricanus* Oberthur. Modern pharmacological studies have shown that Cordyceps sinensis possesses various pharmacological effects, including immunomodulation, hypoglycemia, promotion of hematopoiesis, anti-myocardial ischemia and arrhythmia, antioxidant activity, lipid metabolism regulation, anti-tumor activity, antibacterial activity, and endocrine regulation. In recent years, due to over-harvesting and the destruction of the plateau ecosystem, the yield of wild Cordyceps sinensis has significantly decreased, failing to meet market demand. Therefore, researchers have attempted to artificially cultivate Cordyceps sinensis, that is, to isolate asexual strains of Cordyceps sinensis from wild Cordyceps sinensis, and then ferment these strains to obtain fermented Cordyceps mycelium or powder. *Hirsutella sinensis* is the only known asexual strain of Cordyceps sinensis in China. Modern pharmacological, toxicological, and chemical composition studies have shown that fermented Cordyceps sinensis powder (trade name: Bailin Capsules), produced by low-temperature fermentation of *Hypericum sinense*, possesses effective components and pharmacological effects essentially identical to wild Cordyceps sinensis. It can serve as an artificial substitute for Cordyceps sinensis and be used to treat cough, asthma, hemoptysis, lower back pain caused by deficiency of both lung and kidney, as well as chronic bronchitis and chronic renal insufficiency. Furthermore, existing literature reports that fermented *Hypericum sinense* powder has good anti-inflammatory effects. However, research on the chemical composition of fermented *Hypericum sinense* powder is limited, resulting in a lack of clear understanding of its anti-inflammatory pharmacodynamic material basis.

[0003] Inflammation is a fundamental mechanism of immune defense, protecting against damage caused by harmful stimuli. As pro-inflammatory cytokines, IL-6 and IL-1β are produced by various cells and play important roles in the regulation of inflammation, immune responses, and acute responses. IL-6 is considered a key mediator in the pathogenesis of chronic diseases, playing a role in various pathological inflammatory conditions, including end-stage renal disease and rheumatoid arthritis. Therefore, compounds that regulate the production of IL-1β and IL-6 in LPS-stimulated macrophages have the potential to become novel anti-inflammatory drugs. (III) Summary of the Invention

[0004] The purpose of this invention is to provide a cleaved steroid compound, its preparation method and application. A cleaved steroid compound with good anti-inflammatory activity was obtained from the fermentation powder of *Trichoderma sinense* (the contents of Bailin capsules) through simple extraction and separation.

[0005] The technical solution adopted in this invention is:

[0006] In a first aspect, the present invention provides a cleaved steroidal compound of formula (Ⅰ), also known as pilosporin A, with the molecular formula C. 28 H 44 O5;

[0007]

[0008] Secondly, the present invention provides a method for preparing the aforementioned cleaved steroidal compound, the method comprising the following steps:

[0009] 1) Take the powder of *Hypericum chinense* (the contents of Bailin capsules), extract it with an organic solvent at room temperature, and recover the solvent from the extract under reduced pressure to obtain a crude extract; the organic solvent is 95% ethanol, methanol or acetone, preferably 95% ethanol;

[0010] 2) After suspending the crude extract in water, extract it with an organic solvent, collect the organic solvent phase, and recover the solvent under reduced pressure to obtain the extract; the organic solvent is ethyl acetate, chloroform or dichloromethane, preferably ethyl acetate;

[0011] 3) The extract from step 2) was subjected to gradient elution using a petroleum ether (boiling range 60-90℃) / ethyl acetate mixture at a volume ratio of 30-1:1, at a rate of 50-60 mL / min, with each gradient eluting for 1-5 column volumes. Thin-layer chromatography was performed using dichloromethane / methanol at a volume ratio of 7:1 as the developing solvent. Fractions with an Rf value of 0.4-0.6 were collected. Then, the fractions with an Rf value of 0.4-0.6 were eluted by volume... The fraction with an Rf value of 0.4-0.5 was collected by gradient elution on an MCICHP20P column using methanol / water at a ratio of 60-100:40-0 as the eluent, at a rate of 15-20 mL / min, with each gradient eluting for 1-3 column volumes. Thin-layer chromatography was performed using dichloromethane / methanol at a volume ratio of 7:1. The fraction with an Rf value of 0.4-0.5 was then subjected to gradient elution on an ODS C-18 column using methanol / water at a ratio of 70-100:30-0 as the eluent, at a rate of 10-15 mL / min, with each gradient eluting for 1-3 column volumes. Thin-layer chromatography was performed using dichloromethane / methanol at a volume ratio of 7:1 as the developing solvent. The fraction with an Rf value of 0.5 was collected and concentrated to dryness under reduced pressure to obtain the compound shown in formula (Ⅰ).

[0012] Furthermore, in step 1), the volume of the organic solvent used is 20-50 mL / g (preferably 30 mL / g) based on the weight of the Chinese trichomoniasis fermentation powder; the extraction is performed at least 3 times, with each extraction lasting 3-5 days.

[0013] Further, in step 2), the volume of water used is 2-5 mL / g (preferably 2.3 mL / g) based on the weight of the crude extract; the volume ratio of the organic solvent to water is 1:0.5-2 (preferably 1:3); and the extraction is performed 3-5 times.

[0014] Further, in step 3), the elution conditions for the silica gel column chromatography are as follows: gradient elution is performed sequentially using a petroleum ether / ethyl acetate mixed solvent with a volume ratio of 30:1, 20:1, 10:1, 5:1, 2:1, and 1:1, at an elution rate of 50 mL / min, with each gradient eluting 4 column volumes; the elution conditions for the MCI CHP20P column chromatography are as follows: gradient elution is performed sequentially using a methanol / water mixed solvent with a volume ratio of 60:40, 70:30, 80:20, 90:10, and 100:0, at an elution rate of 15 mL / min, with each gradient eluting 2 column volumes; the ODS The elution conditions for C-18 column chromatography were as follows: gradient elution was performed sequentially using methanol / water mixed solvents with volume ratios of 70:30, 80:20, 90:10, and 100:0, at a elution rate of 10 mL / min, with each gradient elution consisting of 2 column volumes.

[0015] Thirdly, the present invention also provides the use of the aforementioned cleaved steroidal compound in the preparation of anti-inflammatory drugs.

[0016] Furthermore, the anti-inflammatory drug is a drug that inhibits the release of pro-inflammatory cytokines, including IL-6 or IL-1β, particularly inhibiting the release of lipopolysaccharide-induced interleukin-6 (IL-6) and / or interleukin-1β (IL-1β) from macrophages.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] (1) The present invention obtains split-ring steroidal compounds through a series of processes such as organic solvent extraction and chromatographic column separation. The preparation method is simple, rapid and yields compounds with high purity.

[0019] (2) The split-ring steroidal compounds of this invention have good anti-inflammatory activity and can be applied in the development of anti-inflammatory drugs. Using these compounds as lead compounds for structural optimization is of great significance for the development of novel anti-inflammatory drugs. (iv) Description of the attached drawings

[0020] Figure 1 This is the hydrogen spectrum of compound 1.

[0021] Figure 2 This is the carbon spectrum of compound 1.

[0022] Figure 3 This is the DEPT spectrum of compound 1.

[0023] Figure 4 It is compound 1 1 H- 1 H COSY spectrum.

[0024] Figure 5 This is the HSQC spectrum of compound 1.

[0025] Figure 6 This is the HMBC spectrum of compound 1.

[0026] Figure 7 This is the NOESY spectrum of compound 1.

[0027] Figure 8 This is the high-resolution mass spectrum of compound 1.

[0028] Figure 9 This is the infrared spectrum of compound 1.

[0029] Figure 10 The effect of compound 1 on the release of IL-6 and IL-1β from RAW264.1 cells stimulated by LPS. (V) Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0031] The room temperature mentioned in this invention refers to 25-30℃. The *Trichoderma* fermentation powder (contents of Bailin capsules) was purchased from Hangzhou Sino-American East China Pharmaceutical Co., Ltd.

[0032] Example 1: Preparation of Compound 1

[0033] 1. Preparation of crude extract

[0034] Take 1 kg of Chinese spore fermentation powder (the contents of Bailin capsules), extract it 4 times with 30 L of 95% ethanol at room temperature (4 days each time), combine the extracts, concentrate under reduced pressure until no liquid flows out or there is no ethanol odor, and obtain 107 g of crude extract.

[0035] 2. Extraction

[0036] 107g of the crude extract was suspended in 250mL of water and extracted with ethyl acetate, 250mL each time, for a total of 3 extractions; the ethyl acetate phases were combined and the solvent was recovered under reduced pressure to obtain 32g of ethyl acetate extract.

[0037] 3. Separation and purification

[0038] The ethyl acetate extract (32g) was initially separated by silica gel column chromatography (8cm diameter, 50cm height). Gradient elution was performed using petroleum ether (boiling range 60-90℃) / ethyl acetate mixed solvents at volume ratios of 30:1, 20:1, 10:1, 5:1, 2:1, and 1:1 as eluents at a rate of 50mL / min, with each gradient eluting by four column volumes (10L). Thin-layer chromatography (using dichloromethane / methanol 7:1, v / v) was used as the eluent, and fractions with similar Rf values ​​were combined to obtain seven eluted fractions (Fr.A→G). Fr.D (3.1g, Rf value 0.4-0.6) was further separated by MCI CHP. Separation was performed using 20P column chromatography (4 cm diameter, 30 cm height) with gradient elution of methanol / water at volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0 at a rate of 15 mL / min. Two column volumes (800 mL) were eluted for each gradient. Thin-layer chromatography (using dichloromethane / methanol 7:1, v / v) was used as a guide. Fractions with similar Rf values ​​were combined to obtain three elution fractions (Fr.D1→D3). Fr.D2 (72.1 mg, Rf value 0.4-0.5) was then subjected to ODS. Separation was performed using C-18 column chromatography (3.0 cm in diameter and 30 cm in height). Gradient elution was performed using methanol / water at volume ratios of 70:30, 80:20, 90:10, and 100:0 at a rate of 10 mL / min. Two column volumes (250 mL) were eluted for each gradient. Thin-layer chromatography (using dichloromethane / methanol 7:1 as the developing solvent, v / v) was used as a guide. The eluent fraction with an Rf value of 0.5 was collected and concentrated to dryness under reduced pressure to obtain compound 1 (7.4 mg).

[0039] Example 2: Structural Identification of Compound 1

[0040] Compound 1 obtained in Example 1 was subjected to proton, carbon, and DEPT spectra, respectively. 1 H- 1 H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, high-resolution mass spectrometry, and infrared spectroscopy were used for detection. Results are shown in […]. Figures 1-9 Compound 1 is a transparent oil that is readily soluble in methanol.

[0041] (c 0.10, MeOH). According to HRESIMS[M+Na] + m / z:483.3081(C 28 H 44 NaO5 + The calculated value is 483.3081) and 13 C10-NMR data (Table 1) confirmed that the molecular formula of compound 1 is C10-NMR.28 H 44 O5, with an unsaturation degree of 7. It appears at 3386 cm⁻¹ in the infrared spectrum. -1 and 1656cm -1 The absorption bands at these positions indicate the presence of hydroxyl and carbonyl groups in the structure, respectively. The proton NMR spectrum (Table 1) shows six groups of methyl proton peak signals δ... H 1.36 (3H, s), 1.05 (3H, d, J = 7.1 Hz), 1.01 (3H, s), 0.96 (3H, d, J = 6.8 Hz), 0.88 (3H, d, J = 6.8 Hz), 0.86 (3H, d, J = 6.8 Hz), a set of hydroxymethylene proton signals [δ H 3.28 (1H, td, J = 10.2, 6.2 Hz); 3.15 (1H, td, J = 10.2, 5.7 Hz)], three sets of δ-hydroxymethyl proton signals H 4.86 (1H, d, J = 3.0 Hz), 3.79 (1H, m), and 3.18 (1H, d, J = 3.0 Hz), two sets of olefin methylene proton signals δ H 5.42 (1H,dd,J=15.4,8.4Hz), 5.32 (1H,dd,J=15.4,7.8Hz), and methine and methylene proton signals that appear as overlapping signals between chemical shifts of 1.00-2.50ppm. 13 C-NMR and DEPT spectra showed 28 carbon signals, including 6 methyl groups and 7 methylene groups (δ¹²). C 63.8 is the hydroxymethylene carbon), 9 methines (δ) C 61.4, 62.7, and 68.9 are sp. 3 Hybridized hydroxymethyl carbon, δ C 133.9 and 135.9 are SPs. 2 Hybridized olefin methine carbon), seven quaternary carbon atoms (δ C 128.7 and 162.9 are sp. 2 Hybrid olefin quaternary carbon, δ C 205.2 represents a carbonyl group. The above NMR signals indicate that compound 1 contains one carbonyl group and two double bonds, corresponding to three degrees of unsaturation. Therefore, the remaining four degrees of unsaturation indicate that compound 1 has a tetracyclic structure. Furthermore, the above data also show typical characteristics of steroidal compounds, combined with the δ¹⁰ C NMR spectrum... C Signals at 61.4, 63.5, 63.8, 68.9, and 205.2 indicate that compound 1 is a highly oxidized steroid. Careful analysis and comparison revealed that the NMR data of compound 1 are quite similar to those of (22E,24R)-5α,6α-epoxyergosta-8,22-diene-3β,7α-diol, suggesting a structural correlation between the two.1 H- 1 In the HCl COSY spectrum, H2-1 / H2-2 and H2-2 / H-3 (δ) can be observed. H 3.79), H-3 (δ) H 3.79) / H2-4, H-6(δ H 3.18) / H-7(δ H The correlation signals between H2-11 and H2-12 (4.86) suggest the presence of the following three structural fragments in compound 1: -CH2(1)-CH2(2)-CH(3)(OH)-CH2(4)-, -OCH(6)-CH(7)O-, and -CH2(11)-CH2(12)-. In the HMBC spectrum, H3-19 can be observed to correlate with C-1, C-5, and C-9 (δ). C Between 162.9), between H2-4 and C-5, C-6, H-6 (δ) H 3.18) and C-5, C-8 (δ) C Between 128.7) and C-10, H-7 and C-8 (δ) C 128.7), C-9 (δ) C 162.9), C-14 (δ) C Between 205.2), H2-11 and C-8 (δ) C 128.7), C-9 (δ) C Between 162.9) and C-13, H2-12 and C-13, C-14 (δ) C 205.2), H3-18 and C-12, C-13, C-14 (δ C The correlation signals between 205.2) allow the construction of an A / B / C tricyclic system with oxygen bonds at C-3, C-5, C-6, and C-7, and the presence of an Δ8 double bond and a carbonyl group at C-14. The presence of the carbonyl group at C-14 indicates that compound 1 does not possess the five-membered D rings commonly found in ergosterane-type steroids, such as (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7α-diol.

[0042] Furthermore, according to 1 H- 1 H2-15 (δ) in the H COSY spectrum H 3.28 and 3.15) / H-16, H-16 / H-17, H3-21 / H-20, H-20 / H-22 (δ H 5.42), H-22 (δ) H 5.42) / H-23(δ H 5.32), H-23 (δ) HThe related signals of H-24, H-24 / H3-28, H-24 / H-25, H-25 / H3-26, and H-25 / H3-27 can be used to construct the typical side chain CH3(21)-CH(20)-CH(22)=CH(23)-CH(24)(CH3(28))-CH(25)-CH3(26)(CH3(27)) in ergosterane-type steroids, as well as another structural segment HOCH2(15)-CH2(16)-CH(17)-. The HMBC related signals between H-20 and C-16 and C-17 indicate that the above two structural segments are connected through C-17 and C-20, while the HMBC related signal between H3-18 and C-17 indicates that C-17 is connected to C-13, thus constructing a 14,15-secrosor ergosterane skeleton. By comparing the chemical shifts of C-5, C-6, and C-7 in compound 1 and (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7α-diol, and combining this with the molecular formula of compound 1, it can be determined that C-5 and C-6 of compound 1 form an epoxide. Thus, the planar structure of compound 1 has been determined, and this compound possesses a rare 14,15-sec-cyclic ergoster skeleton.

[0043] From a biogenic perspective, compound 1 is mainly produced by the oxidative cleavage of the Δ14 double bond of (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7α-diol. This reaction process does not involve changes to existing chiral centers in the molecule, nor does it generate new chiral centers. Therefore, the corresponding chiral centers in the two compounds should have the same relative configuration. This inference was also verified by resolving NOESY spectra, analyzing coupling constants, and comparing carbon spectral chemical shifts. In ergosterane-type steroids, the two angular methyl groups of Me(18) and Me(19) are in the β configuration. In NOESY spectra, H3-19 and H can be observed. a NOE-related signals between -4 indicate H a -4 represents the β orientation. Therefore, H-3 / H b The NOE correlation signal between -4 indicates that H-3 is α-oriented. The NOE signal between H-6 / H-7 / H3-19 indicates that both 5,6-epoxy and OH-7 are α-oriented. Furthermore, H-20 / H... a The NOE correlation signal between -12 / H3-18 suggests that the C-17 side chain is β-oriented. The large coupling constant between H-22 and H-23 (… 3 J H-22,H-23=15.4 Hz) indicates that the Δ22 double bond has an E configuration. Furthermore, by comparing the carbon chemical shifts of the side chains of compound 1 and (22E)-5α,6α-epoxyergosta-8,14,22-triene-3β,7α-diol, it can be determined that the C-20 and C-24 positions of the two compounds have the same configuration. Based on the above analysis, compound 1 is resolved to have the structure shown in formula (Ⅰ) and named trichosporine A.

[0044]

[0045] Formula (I) represents the structure of compound 1; Formula (II) represents the main structure. 1 H- 1 H COSY and HMBC are correlated; Equation (III) is the main NOE correlation.

[0046] Table 1. NMR data of compound 1 (CDCl3; 1H and 1C spectra measured at 600 and 150 MHz, respectively).

[0047]

[0048]

[0049] Example 3: Evaluation of the in vitro anti-inflammatory activity of compound 1

[0050] 1. Macrophage RAW264.7 culture

[0051] After resuscitation, RAW264.7 macrophages (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded into Dulbecco's modified eagle medium (DMEM) high-glucose medium containing 10% fetal bovine serum (FBS) and incubated in an incubator at 37°C, 5% CO2, and 90% relative humidity. During culture, care should be taken to avoid excessively rapid cell growth and high cell density; subculturing and medium changes should be performed when the cell density reaches 80-90% to prevent long-tentacle polarization. RAW264.7 cell subculturing does not require trypsin digestion; cells are gently pipetted using a dropper and passaged at a ratio of 1:2.

[0052] 2. RT-qPCR quantitative PCR method for measuring inflammatory factors IL-6 and IL-1β

[0053] 2.1 Cell Culture and Drug Dosing Methods

[0054] RAW264.7 cells in logarithmic growth phase were seeded at 200,000 cells / well in 12-well plates and cultured at 37°C in a 5% CO2 cell culture incubator until the cell density reached 80%. A control group, a lipopolysaccharide (LPS) inflammation model group, a positive control group (dexamethasone), and a sample group were set up. The LPS inflammation model group was treated with LPS at a final concentration of 1 μg / mL. The sample groups were treated with compound 1 at final concentrations of 1, 3, and 9 μM, respectively, in addition to the treatment given to the model group. The positive control group was treated with dexamethasone at final concentrations of 1, 3, and 9 μM, respectively. The control group received no treatment. Cells were cultured for 12 hours. The culture medium was discarded, and the cells were collected for subsequent experiments.

[0055] 2.2 Total RNA extraction from cells

[0056] Place the 12-well plate (without culture medium) on ice. Add 0.5 mL of Trizol lysis buffer to each well and lyse for 10 min. Pipettes the cells using a pipette tip and transfer them to a 1.5 mL centrifuge tube. Incubate for 5 min, then add 200 μL of chloroform and vortex until pink. Incubate for 5 min and centrifuge at 12000 rpm, 4°C for 15 min. Carefully collect 100 μL of the supernatant (aqueous phase) and transfer it to a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol, mix well, incubate for 10 min, and centrifuge at 12000 rpm, 4°C for 10 min. A small amount of white gelatinous substance can be observed at the bottom of the centrifuge tube at this point. Discard the supernatant, add 1 mL of 75% ethanol, shake well to suspend the precipitate, incubate for 5 min, and centrifuge at 12000 rpm, 4°C for 5 min. Discard the supernatant and dry at room temperature for 10 min to obtain RNA. Add 15 μL of sterile, enzyme-free water to fully dissolve the RNA, and measure the RNA concentration using a Thermo Nanordrop 2000 nucleic acid and protein analyzer.

[0057] 2.3 Synthesis of cDNA

[0058] Following the instructions for the TaKaRa reverse transcription kit, the synthesized cDNA can be used directly for qPCR or stored at -80°C for later use.

[0059] 2.4 RT-qPCR reaction

[0060] 20 μL qPCR reaction system: 10 μL Hieff qPCR SYBR Green Master Mix, 0.4 μL Forward primers, 0.4 μL Reverse primers, 8.2 μL sterile enzyme-free water, and 1 μL cDNA of the sample to be tested. Mix thoroughly. Real-Time PCR reaction conditions were set to 95℃ for 5 min pre-denaturation, followed by 40 cycles of 95℃ for 10 s and 60℃ for 30 s. Using 2...-△△Ct Analysis results, △ Ct =Ct (目的基因) -Ct (内参基因GAPDH) , △△ Ct =△ Ct(实验组) -△ Ct(对照组) .

[0061] Table 2. Specific primers for RT-qPCR

[0062]

[0063] 3. Results of anti-inflammatory activity evaluation experiments

[0064] The effect of compound 1 on the release of inflammatory cytokines IL-1β and IL-6 from LPS-stimulated RAW 264.7 cells was determined using qPCR. The results are as follows: Figure 10 As shown, the expression levels of IL-6 and IL-1β mRNA in the model group were 117.7 and 13.8 times higher than those in the control group, respectively, indicating that the inflammation model was successfully established. In the positive control group (1 μM) of dexamethasone, the relative expression levels of IL-6 and IL-1β mRNA were significantly lower than those in the model group, decreasing by 73.4% and 89.3%, respectively. Low (1 μM), medium (3 μM), and high (9 μM) doses of compound 1 reduced the expression level of IL-6 mRNA by 14.0%, 24.2%, and 45.0%, respectively, and reduced the relative expression level of IL-1β mRNA by 23.4%, 35.0%, and 57.6%, respectively, indicating that compound 1 can reduce the LPS-induced inflammatory response in RAW 264.7 cells in a dose-dependent manner.

[0065] The above results indicate that compound 1 involved in this invention has good anti-inflammatory effects and is expected to be developed into a novel anti-inflammatory drug.

Claims

1. A cleaved steroid compound of formula (Ⅰ), 2. A method for preparing the cleaved steroid compound of claim 1, characterized in that, The method includes the following steps: 1) Take the fermentation powder of *Hypericum chinense* from China, extract it with an organic solvent at room temperature, and recover the solvent from the extract under reduced pressure to obtain a crude extract; the organic solvent is 95% ethanol, methanol or acetone. 2) After suspending the crude extract in water, extract it with an organic solvent, collect the organic solvent phase, and recover the solvent under reduced pressure to obtain the extract; the organic solvent is ethyl acetate, chloroform or dichloromethane; 3) The extract from step 2) was subjected to gradient elution using a petroleum ether / ethyl acetate mixture (v / v) at a ratio of 30–1:1 at silica gel column chromatography at a rate of 50–60 mL / min, with each gradient eluting for 1–5 column volumes. Thin-layer chromatography (TLC) was performed using dichloromethane / methanol (v / v) at a ratio of 7:1, and fractions with an Rf value of 0.4–0.6 were collected. Then, the fractions with an Rf value of 0.4–0.6 were subjected to MCI elution using a methanol / water mixture (v / v) at a ratio of 60–100:40–0. Gradient elution was performed on a CHP20P column at a rate of 15-20 mL / min, with each gradient eluting for 1-3 column volumes. Thin-layer chromatography was then performed using dichloromethane / methanol at a volume ratio of 7:1, and fractions with an Rf value of 0.4-0.5 were collected. The fractions with an Rf value of 0.4-0.5 were then subjected to gradient elution on an ODS C-18 column at a rate of 10-15 mL / min, with each gradient eluting for 1-3 column volumes. Thin-layer chromatography was then performed using dichloromethane / methanol at a volume ratio of 7:1, and fractions with an Rf value of 0.5 were collected. The mixture was concentrated to dryness under reduced pressure to obtain the compound shown in formula (Ⅰ).

3. The preparation method according to claim 2, characterized in that, In step 1), the volume of the organic solvent used is 20-50 mL / g based on the weight of the Chinese spore fermentation powder.

4. The preparation method according to claim 2, characterized in that, In step 2), the volume of water used is 2-5 mL / g based on the weight of the crude extract; the volume ratio of the organic solvent to water is 1:0.5-2.

5. The preparation method according to claim 2, characterized in that, In step 3), the elution conditions for silica gel column chromatography are as follows: gradient elution is performed sequentially using a petroleum ether / ethyl acetate mixed solvent with volume ratios of 30:1, 20:1, 10:1, 5:1, 2:1, and 1:1, with each gradient elution consisting of 4 column volumes.

6. The preparation method according to claim 2, characterized in that, In step 3), the elution conditions for the MCI CHP20P column chromatography are as follows: gradient elution is performed sequentially using methanol / water mixed solvents with volume ratios of 60:40, 70:30, 80:20, 90:10, and 100:0, with each gradient elution consisting of 2 column volumes.

7. The preparation method according to claim 2, characterized in that, In step 3), the elution conditions for the ODS C-18 column chromatography are as follows: gradient elution is performed sequentially using methanol / water mixed solvent with volume ratios of 70:30, 80:20, 90:10, and 100:0, with each gradient elution consisting of 2 column volumes.

8. The use of the split-ring steroid compound of claim 1 in the preparation of an anti-inflammatory drug.

9. The application as described in claim 8, characterized in that, The anti-inflammatory drug is a drug that inhibits the release of pro-inflammatory cytokines, including IL-6 or IL-1β.

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